There is a strong need for rapid and simplified upstream processing of complex samples for (bio)chemical analysis. This need is common to conventional laboratory and diagnostic assay systems, as well as to newer microdevice and microfluidic systems that offer simplified fluid handling, conservation of scarce samples and reagents, and portability.
The controlled separation and release of specific biological molecules from complex mixtures is a key process in most bioanalytical and diagnostic technologies. Efficient, specific bioseparation processes are necessary because of the stringent purity requirements for many bioanalytical and diagnostic technologies. Affinity bioseparation systems rely on a specific biological interaction between a target biomolecule and an affinity ligand. Affinity chromatography is the most commonly used affinity bioseparation technology that relies on the attachment of an affinity ligand to an immobile matrix. A complex mixture containing the target biomolecule is passed over this matrix, and the target molecule binds to its affinity ligand, allowing the other components of the mixture to be washed away. The separated target molecule is then eluted from the column, typically with a chemical eluent that weakens the affinity interaction. Affinity chromatography is very efficient and specific in purifying target biomolecules and cells.
Affinity separations typically rely on biomolecular recognition. The control of recognition steps is thus an integral aspect of bioprocessing steps that accompany bioanalytical and diagnostic technology. For many affinity separation, diagnostic, biosensor, biochip and bioprocessing technologies that utilize biomolecular recognition properties, there is a continuing need for better control routes. Many of the current methods are relatively harsh and can lead to damage of biomolecules and cells. In addition, the environmental signals are typically large general solution changes and thus not targeted to selective recognition components.
“Smart” polymers, which reversibly change their physical properties in response to small and controllable stimuli (e.g., changes in pH, temperature, and light), to control recognition events by acting as environmental antennae and switches. These smart polymers reversibly cycle between an extended and hydrophilic random coil, and a collapsed, hydrophobic state that is reduced in average volume by about 3-fold. The polymers serve as environmental sensors and differentially control access of ligands or substrates to binding or catalytic sites as a function of their expanded or collapsed states. This general approach targets mild environmental signals to specific polymer-protein conjugates, and thus, for example, allows differential control of different antibodies in a device by using conjugated polymers that are sensitive to different signals (e.g., antibody 1 with pH, antibody 2 with temperature, antibody 3 with light).
The following references describe various efforts to employ affinity recognition for the controlled separation and release of bioanalytical, therapeutic drug, or diagnostic agents. References implementing the smart polymers mentioned above are included.
U.S. Pat. No. 5,451,411 describes alginate beads designed to deliver cationic therapeutic agents to the luminal side of the small intestine via oral ingestion. Co-encapsulation of polyanionic additives with the therapeutic agent followed by acid treatment of the resulting bead enhances release of the agent. Sustained release of the agent is triggered within a nontoxic gastrointestinal pH range.
U.S. Pat. No. 5,770,627 describes hydrophobically-modified bioadhesive polyelectrolytes capable of sustained release of a pharmaceutically, cosmetically, or prophylactically acceptable agent. The hydrophobic component (a hydrophobic moiety or hydrophobic polymer) facilitates micelle formation, permitting delivery of the cationic or hydrophobic and/or anionic agent. Uptake of water by the bioadhesive polyelectrolyte portion (a carboxylic acid-containing polymer) results in swelling and “stickiness,” thereby allowing the sustainable release of the agent.
U.S. Pat. No. 6,486,213 describes block and graft copolymers for use in the topical delivery of drugs. The copolymer is physically mixed with one or more drugs to form a copolymer-drug mixture. These copolymers contain a pH-sensitive polymer component, which swells and adheres to the treatment area upon uptake of water, and a temperature-sensitive polymer component, which facilitates controlled release of the drug.
U.S. Pat. No. 5,998,588 describes stimuli-responsive interactive molecular conjugates. The conjugates include a stimuli-responsive component that is an environmentally sensitive polymer and an interactive molecular component that can be a variety of ligand-binding biomolecules. In the conjugate, a stimuli-responsive polymer is coupled to an interactive biomolecule proximal to the ligand-binding site of the ligand-binding molecule such that, upon stimulation, the polymer alters ligand-biomolecule binding behavior. External stimuli, such as temperature, pH, or light cause the stimulus-responsive component to undergo a conformational or physico-chemical change that can lead to a structural transition in the conjugate itself, thereby modulating the activity of the interactive biomolecule.
U.S. Pat. No. 6,165,509 describes PEGylated drugs complexed with bioadhesive polymers (e.g., polyacrylic, polymethacrylic, polyethylacrylic acids, and chitosan). The PEGylated drug includes a polyethylene glycol covalently bonded to the drug. Upon uptake of water at the treatment site, the bioadhesive polymer becomes “sticky” and neutral pH exposure facilitates the dissociation of the PEGylated drug from the bioadhesive polymer. Sustained drug release is thereby achieved via topical administration.
WO 01/51092 describes a composition, for disruption of cell membrane, used for delivering diagnostic or therapeutic agents to cytoplasm of cells. The composition includes a conjugate having a hydrophobic component linked to a hydrophilic component by a linkage capable of being disrupted or degraded, preferably by a change in pH. The conjugate can further include a therapeutic, diagnostic, or prophylactic agent. The hydrophobic component (e.g., environmentally sensitive polymer) is membrane disruptive. The hydrophilic conjugate operates to first enhance membrane transport of the agent, to next undergo linkage degradation, and to finally release of the agent into the cytosol.
Despite the advances noted above, there still exists a need for improved methods of biomolecule capture and release. The present invention seeks to fulfill this need and provides further related advantages.